Field of the Invention
The present invention relates to a method for producing an acidic substance having a carboxyl group. L-Glutamic acid and L-aspartic acid are widely used as raw materials in making seasonings and so forth. Succinic acid is widely used as a raw material in making seasonings and biodegradable plastics.
Brief Description of the Related Art
L-Glutamic acid is mainly produced by fermentation utilizing L-glutamic acid-producing bacteria of the so-called coryneform bacteria belonging to the genus Brevibacterium, Corynebacterium or Microbacterium, or their mutant strains (see, for example, Kunihiko Akashi et al., Amino Acid Fermentation, Japan Scientific Societies Press (Gakkai Shuppan Center), pp. 195-215, 1986). Methods are known for producing L-glutamic acid by fermentation using other bacterial strains, including microorganisms belonging to the genera Bacillus, Streptomyces, Penicillium or the like (see, for example, U.S. Pat. No. 3,220,929), microorganisms belonging to the genera Pseudomonas, Arthrobacter, Serratia, Candida or the like (see, for example, U.S. Pat. No. 3,563,857), microorganisms belonging to the genera Bacillus, Pseudomonas, Serratia, Aerobacter aerogenes (currently referred to as Enterobacter aerogenes) or the like (see, for example, Japanese Patent Publication (KOKOKU) No. 32-9393), a mutant strain of Escherichia coli (see, for example, Japanese Patent Laid-open (KOKAI) No. 5-244970), and the like. In addition, methods for producing L-glutamic acid have also been disclosed using microorganisms belonging to the genera Klebsiella, Erwinia, Pantoea or Enterobacter (see, for example, Japanese Patent Laid-open No. 2000-106869 (U.S. Pat. No. 6,682,912), Japanese Patent Laid-open No. 2000-189169 (U.S. Patent Published Application No. 2001009836), Japanese Patent Laid-open No. 2000-189175 (U.S. Pat. No. 7,247,459)).
Furthermore, various techniques have been disclosed for increasing the L-glutamic acid-producing ability by enhancing the L-glutamic acid biosynthetic enzymes using recombinant DNA techniques. For example, it has been reported for Corynebacterium or Brevibacterium bacteria that introduction of a gene coding for citrate synthase derived from Escherichia coli or Corynebacterium glutamicum was effective to enhance the L-glutamic acid-producing ability of coryneform bacteria (see, for example, Japanese Patent Publication No. 7-121228). Furthermore, it has also been reported that introduction of a citrate synthase gene derived from a coryneform bacterium into enterobacteria belonging to the genera Enterobacter, Klebsiella, Serratia, Erwinia or Escherichia was effective to enhance the bacteria's L-glutamic acid-producing ability (see, for example, Japanese Patent Laid-open No. 2000-189175 (U.S. Pat. No. 7,247,459)).
Methods for improving the production of target substances such as amino acids are also known, including by modifying the uptake or secretion systems of target substances. Such methods include, for example, by deleting or attenuating the system for uptake of a target substance into cells. Specifically, known methods to improve production of L-glutamic acid include deleting the gluABCD operon, or a part thereof, to eliminate or attenuate uptake of L-glutamic acid into cells (see, for example, European Patent Application Laid-open No. 1038970), and enhancing production of purine nucleotides by attenuating uptake of purine nucleotides into cells (see, for example, European Patent Application Laid-open No. 1004663), and the like.
Furthermore, methods of enhancing the secretion system for a target substance, and methods of deleting or attenuating the secretion system for an intermediate or substrate in the biosynthetic system of a target substance are known. Known methods of enhancing the secretion system of a target substance include, for example, production of L-lysine by utilizing a Corynebacterium strain in which the L-lysine secretion gene (lysE) is enhanced (see, for example, WO2001/5959), and production of L-glutamic acid by using an enterobacterium in which the L-glutamic acid secretion system gene (yhfK) is enhanced (see, for example, Japanese Patent Laid-open No. 2005-278643 (U.S. Patent Published Application No. 2005196846)). Furthermore, methods for producing an L-amino acid using the rhtA, B, C genes, which have been suggested to be involved in the secretion of L-amino acids, have also been reported (see, for example, Japanese Patent Laid-open No. 2000-189177 (U.S. Patent Published Application No. 2005239177)). Known methods for, for example, deleting a secretion system for an intermediate or substrate in a biosynthesis system of a target substance include, for L-glutamic acid, mutating or disrupting the 2-oxoglutarate permease gene to attenuate secretion of 2-oxoglutarate, which is an intermediate of the target substance (see, for example, WO97/23597).
Furthermore, use of the gene coding for the ATP binding cassette superfamily (ABC transporter), which is involved in transportation of substances through cell membranes, in the breeding of microorganisms in which transmembrane transportation of amino acids is modified has been suggested (see, for example, WO00/37647).
Furthermore, it has also been reported that L-glutamic acid production efficiency can be improved in Escherichia bacteria by enhancing the expression of genes thought to participate in secretion of L-amino acids such as yfiK (see, for example, Japanese Patent Laid-open No. 2000-189180 (U.S. Pat. No. 6,979,560)). Moreover, it has also been reported that L-glutamic acid-producing ability can be improved by enhancing expression of the yhfK gene (see, for example, Japanese Patent Laid-open No. 2005-278643 (U.S. Patent Published Application No. 2005196846)).
Moreover, methods are known for producing L-glutamic acid by culturing a microorganism under acidic conditions to precipitate the L-glutamic acid (see, for example, Japanese Patent Laid-open No. 2001-333769 (U.S. Patent Published Application No. 2007134773)). When the pH is kept low, L-glutamic acid is precipitated, and the ratio of L-glutamic acid in free form with no electrical charge increases. As a result, the L-glutamic acid easily penetrates cell membranes. When L-glutamic acid is taken up into cells, it is converted into an intermediate of the TCA cycle, 2-oxoglutaric acid, in one step by glutamate dehydrogenase, and therefore it is generally thought that L-glutamic acid taken up into cells is easily metabolized. However, 2-oxoglutarate dehydrogenase activity can be deleted or attenuated, or the like, in the fermentative production of L-glutamic acid (for example, Japanese Patent Laid-open No. 2001-333769 (U.S. Patent Published Application No. 2007134773), Japanese Patent Laid-open No. 7-203980 (U.S. Pat. No. 5,573,945)), but then 2-oxoglutaric acid is not degraded, intracellular 2-oxoglutaric acid concentration increases which inhibits the growth of the microorganism. Thus, the culture fails. Therefore, as described in Japanese Patent Laid-open No. 2001-333769 (U.S. Patent Published Application No. 2007134773), a strain was bred using a mutation that is deficient in 2-oxoglutarate dehydrogenase activity and can produce L-glutamic acid accompanying precipitation, and this strain can be used for the production of L-glutamic acid.
For fermentative production of non-amino organic acids, including succinic acid, anaerobic bacteria including those belonging to the genus Anaerobiospirillum or Actinobacillus are usually used (U.S. Pat. Nos. 5,142,834 and 5,504,004, Guettler, M. V. et al., 1999, International Journal of Systematic Bacteriology, 49:207-216). Although the use of such anaerobic bacteria provides high product yields, many nutrients are required for sufficient proliferation, and therefore, it is necessary to add large amounts of organic nitrogen sources such as corn steep liquor (CSL) into the culture medium. The addition of large amounts of organic nitrogen sources can result in not only an increase in the cost of the culture, but also an increase in the cost for isolating or purifying the product, and therefore, their use is not economical.
In addition, methods are known in which aerobic bacteria such as coryneform bacteria are cultured once under aerobic conditions, then harvested, washed, and allowed to rest, producing a non-amino organic acid in the absence of supplied oxygen (Japanese Patent Laid-open Nos. 11-113588 and 11-196888). These methods are economical since a smaller amount of organic nitrogen can be added, and the bacteria will sufficiently grow in a simple culture medium. However, there is still a room for improvement in terms of production amount, concentration, and production rate per cell of the target organic acids, as well as simplification of the production process, and the like. Furthermore, the production of a non-amino organic acid by fermentation using a bacterium in which phosphoenolpyruvate carboxylase activity is enhanced (for example, Japanese Patent Laid-open No. 11-196887), and the like have also been reported.
Furthermore, as for Escherichia coli, which is a facultative anaerobic gram negative bacterium, methods for producing a non-amino organic acid by culturing it once under aerobic conditions, and then allowing the cells to rest in the absence of supplied oxygen, resulting in an anaerobically produced non-amino organic acid (Vemuri G. N. et al., 2002, Journal of Industrial Microbiology and Biotechnology, 28(6):325-332). This is similar to the methods using coryneform bacteria. Alternatively, E. coli can be aerobically cultured to aerobically produce the non-amino organic acid (U.S. Patent Published Application No. 20050170482). However, since Escherichia coli is a gram negative bacterium, it is vulnerable to osmotic pressure, and there remains room for improvement in productivity per cell etc.
The ybjL gene is located on the genome of Escherichia coli (see, for example, Blattner, F. R. et al., 1997, Science, 277(5331):1453-74), and it is also thought to code for a transporter on the basis of the motifs, topology etc. of the deduced amino acid sequence. However, cloning of the gene, as well as expression of the gene and analysis of the expression product have not been reported, and the actual functions of the gene remained unknown.